Hamf12

H. capsulatum strains used in this study included G186A (ATCC 26029), representative of chemotype II, and EH-315. EH-315 was isolated from the intestine of infected bats captured in a cave in the state of Guerrero (Mexico) and is designated by Teixeira et al. (2016) (link) as belonging to a bat-associated species-specific clade (BAC1). EH-315 is deposited in the H. capsulatum Culture Collection of the Fungal Immunology Laboratory of the Department of Microbiology and Parasitology, from the School of Medicine, National Autonomous University of Mexico (UNAM) (www.histoplas-mex.unam.mx), which is registered in the database of the World Data Centre for Microorganisms (WDCM) with number LIH-UNAM WDCM817. The G186A is classified as H81 human lineage (Kasuga et al., 2003 (link)). Both strains are now deposited in the collection of strains at the Clinical Mycology Laboratory of the Faculty of Pharmaceutical Sciences, UNESP (Brazil), and maintained at 37°C in Brain Heart Infusion agar supplemented with 1% of glucose and 0.1% of L-cysteine. Before the experiments, H. capsulatum was cultivated in Histoplasma-macrophage medium (HMM), composed of HAM-F12 (Sigma) medium, supplemented with glucose (18.2 g/L), glutamic acid (1g/L), HEPES (6 g/L), and L-cysteine (8.4 mg/L) at 37°C and 150 rpm for 48 h.

BCLC9 cells were generated as previously described¹². Standard culture medium for BCLC9 cell lines is Dulbecco's Modified Eagle Medium (DMEM) high glucose (Sigma-Aldrich, St Louis, MO) and HAM F12 (Sigma-Aldrich, St Louis, MO) (1:1). Medium is supplemented with: 1% Na Pyruvate 100 mM (Sigma-Aldrich, St Louis, MO), 1% Pen/Strep 10000 U/mL (Lonza Group Ltd, Switzerland), 1% L-Gln 200 mM (Sigma-Aldrich, St Louis, MO), 1% Non-Essential Aminoacids (NEAA) (Lonza Group Ltd, Switzerland), 10% FBS (Life Technologies). BCLC9-miR122 and BCLC9-AKT3 KD culture medium is supplemented with neomycin (G418 disodium salt, Sigma-Aldrich, Germany; at a final concentration of 500 μgr/mL) and puromycin (Sigma-Aldrich, Germany; at a final concentration of 10 μgr/mL) respectively, once a week to keep selective pressure.

ITSCs were isolated from adult human inferior turbinate tissue obtained by biopsy during routine surgery after informed consent according to local and international guidelines. The cells were expanded within the 3D plasma matrix as described previously [6 (link),58 (link)], cultivated in Dulbecco’s modified Eagle’s medium/Ham F-12 (Sigma-Aldrich, St. Louis, MO, USA) supplemented with basic fibroblast growth factor-2 (FGF2; 40 ng/mL; Miltenyi Biotec, Bergisch Gladbach, Germany), epidermal growth factor (EGF; 20 ng/mL; Miltenyi Biotec) and B27 (Gibco) followed by supplementation with 10% of clinically accredited therapeutic human plasma (obtained from Institut für Laboratoriums- und Transfusionsmedizin, Bad Oeynhausen, Germany) and cultivated at 37 °C, 5% O₂ and 5% CO₂. All experimental procedures were ethically approved by the ethics board of the medical faculty of the University of Münster (No. 2012–015-f-S).

HaCaT and DOK cells were cultured in DMEM (Dulbecco’s Modified Eagle’s Medium)/Ham F12 (Sigma-Aldrich Chemie Gmbh, Munich, Germany) and supplemented with 10% fetal bovine serum (FBS), and antibiotic-antimycotic solution (both from Lonza, Basel, Switzerland). Cells were seeded in 35 mm glass bottom petri dishes (7.5 × 10⁵ cells/dish), for time-lapse videomicroscopy wound healing experiments, or in 16 wells E-plates (7.5 × 10³ HaCaT cells/well, and 15 × 10³ DOK cells/well, respectively) for time-lapse impedance monitoring. To prepare culture surfaces for the experiments, three different matrix proteins were used: collagen type I, from calf skin (C9791, Sigma-Aldrich Chemie Gmbh); fibronectin from bovine plasma (33010-018, Invitrogen Molecular Probes, Eugene, OR, USA); and laminin from mouse basement membrane (354232, BD Bioscience, San Jose, CA, USA).

BeWo and JEG-3 cell lines were maintained at standard culture conditions of 5% CO₂ in air at 37°C. BeWo cells were cultured in Ham F12 (Sigma-Aldrich, UK) containing 10% heat-inactivated fetal bovine serum (FBS) and 0.5% penicillin streptomycin, whereas JEG-3 cells were maintained in MEME (Sigma-Aldrich, UK) containing 10% heat-inactivated FBS and 0.5% penicillin streptomycin, 0.5% L-glutamine, 0.5% sodium pyruvate, and 0.5% MEM nonessential amino acids. Prior to cortisol treatment, both cell lines were maintained for 3 hours in phenol red-free media containing charcoal stripped FBS.